The presence of multiple prion strains is a great challenge to the prion hypothesis, which postulates that the variations in the pathogenic PrP conformation, PrPSc, lead to the prion strain phenomenon. Recent advance in prion field reveals a key role of other physiological factor(s) in facilitating PrP conversion and generating prion infectivity. Our recent results showed that, in the presence of proper facilitating factors, the bacterially-expressed recombinant PrP can be converted into the infectious conformation causing bona fide prion disease in animals. This discovery strongly supports the prion hypothesis, highlights the important role of facilitating factors in prion conversion, and opens new avenues for prion research. We propose to use the de novo recombinant prion formation system to study the role of facilitating factors in enciphering prion strains. We will use the in vitro PrP conversion assay, biochemical characterization, animal bioassay, and histopathological analyses to investigate the relationship among facilitating factors, the infectious prion aggregates, and the prion strain phenomenon in the following three specific aims. In aim 1, we propose to determine whether transmission in outbred mice with a single recombinant prion preparation is capable of creating multiple prion strains. In aim 2, we will determine whether two biochemically different recombinant prions represent two different strains. In aim 3, we will test the hypothesis that distinct prion strains can be created in the presence of different facilitating factors. Results from these studies will provide us with insights into the molecular mechanism behind prion strain phenomenon, which is critically important in preventing cross-species transmission of these fatal neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Prion diseases are a group of infectious neurodegenerative diseases affecting both human and animals. The most important issue in prion research is to prevent the potential spread of prion disease from animal to humans. Prion strain phenomenon is intimately related to the cross species transmission, and therefore, our proposed study is highly relevant to human health. Furthermore, the knowledge generated from proposed studies may also lead to new strategies against these devastating and incurable diseases.